Combination of Polymeric Microneedles with Specific Peptide for TNF-α Sensing Via Noninvasive Detection in Interstitial Fluid

Abstract Microneedle (MN)-based biosensors offer a minimally invasive and blood-free approach for real-time monitoring of clinically relevant biomarkers in interstitial fluid (ISF). Pro-inflammatory cytokines such as Tumor Necrosis Factor-α (TNF-α) can reach higher concentrations in ISF than in serum, which makes this fluid particularly sensitive for detecting early local inflammatory responses. Here, we introduce an antibody-free MN biosensor employing a synthetic high-affinity peptide (P52) for TNF-α recognition in ISF. The novelty of the platform lies in the stable immobilization of the P52 peptide directly on mechanically robust polymeric MNs for a scalable three-dimensional fluorescence assay. The results reported show how the limitations commonly associated with translating monoclonal antibody-based molecular recognition could be overcome using a functionalized polymeric microneedle platform. Poly(lactic-co-glycolic acid) MNs were fabricated via micro-molding and subsequently functionalized with the P52 peptide. A simple surface-conjugation protocol was employed, which assured peptide stability, low production costs, and reliable conjugation. Comprehensive morphological, mechanical, and chemical characterizations were performed through SEM and fluorescence microscopy. Quantitative detection was achieved via a sandwich-type fluorescence assay using an FITC-labeled P52 variant, enabling evaluation of capture efficiency in simulated ISF across graded TNF-α concentrations. The platform exhibited a linear response with a limit of detection of ∼0.2 pM (∼3 pg/mL). Peptide immobilization on MNs enhanced binding affinity, shifting the equilibrium dissociation constant from the nanomolar to the picomolar range. The P11 peptide was used as a negative control for sequence-dependent TNF-α recognition. Mechanical testing confirmed the robustness of the functionalized MNs without structural failure. Overall, this peptide-functionalized MN platform represents a robust, sensitive, and scalable solution for point-of-care TNF-α monitoring directly in ISF, paving the way for next-generation minimally invasive and low-cost diagnostic systems that will revolutionize the remote biomedicine solution.